UCSF

The Notch signaling pathway is required in sensing the local cellular environment during multicellular development. Because Notch utilizes juxtacrine contact between two cells and relies upon a set of protease cleavage events, understanding the distribution and dynamics of the receptor during activation are of particular importance in order to understand its regulation. Notch must not just be expressed and present at the cell surface, but must be at the right place at the right time for proper activation to occur. Here we invent and then use monovalent Quantum Dots (mQDs) to target and monitor human Notch1 on the surface of live mammalian cells. We track and observe the dynamics of the receptor and compare the diffusion and distribution of Notch with other structural features at the surface of the cell. We find Notch to be slow and confined in its diffusion at the cell surface. In comparing temporal dynamics with static high-resolution static microscopy of the receptor we find Notch to be excluded from particular regions on the surface. These regions of exclusion include focal adhesions as determined by co-imaging Paxillin and Notch. In an attempt to determine the mechanism of exclusion we systematically pared domains from the receptor and identify the extracellular Notch regulatory region (NRR) as playing a significant role in distributing the receptor at the cell surface.

Additional work tries to quantitate the emergent behaviors observed in intercellular interactions taking place in heterogeneous collections of tissues. We repurpose single molecule tracking techniques used to track single molecules in order to track and identify the distribution and dynamics of cells to arrive at quantitative descriptions of multicellular interactions. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.